Project Summary Although smallpox had been eradicated, the chance the virus could arise from unknown stocks or de novo synthesis remains a credible biothreat. Moreover, other poxviruses cause significant morbidity and mortality in humans and economically important animals; and poxviruses are widely being developed as vaccine and protein expression vectors, and anti-cancer agents. Poxvirus gene expression is an arguably irreplaceable area in elucidating fundamental mechanisms of poxvirus replication and developing poxvirus-based utilities. Vaccinia virus (VACV) is the prototype poxvirus. VACV post-replicative genes (those expressed after viral DNA replication) number over 90 and encode mainly virion-associated proteins. They are more efficiently translated than cellular mRNAs and expressed at high levels. The objective of this project is to elucidate molecular mechanisms driving selective translation of VACV post-replicative mRNAs, all of which have an unusual feature?a 5'-poly(A) leader. During the post-replicative stage of VACV infection, the poly(A) leader appears to confer a translational advantage. The central hypothesis of this project is that the 5'-poly(A) leader coordinates with specific viral and cellular factors to promote translation of VACV post-replicative mRNAs. Supporting this hypothesis, D9 and D10, two VACV-encoded decapping enzymes that are known to promote cellular and viral mRNA degradation, are required for efficient translation of VACV post-replicative mRNAs. Exogenous expression of D10 in uninfected cells promotes translation of mRNA with a 5'-poly(A) leader. This is striking given that activation of cellular decapping enzymes causes translation repression. Moreover, La-related protein 4 (LARP4), a cellular poly (A)-binding protein and mRNA translation regulator, is required for VACV post- replicative protein synthesis. LARP4 is also highly enriched in viral factories, where viral post-replicative mRNA translation occurs. Three Specific Aims will be carried out to test how these factors promote selectively translation of VACV post-replicative mRNAs. In Aim 1, the role and molecular determinants of poxvirus decapping enzymes in promoting viral post-replicative mRNA translation will be defined. In Aim 2, the mechanisms by which VACV decapping enzymes promote viral post-replicative mRNA translation will be elucidated. In Aim 3, how LARP4 regulates VACV post-replicative mRNA translation will be determined. The results will provide an integrated new paradigm illustrating how an intrinsic sequence in viral transcripts, specific viral and cellular factors orchestrate selective translation of VACV post-replicative mRNAs, and in turn, VACV replication. The information can be used to tune or block poxvirus replication and improve the efficacy of VACV vectors used in vaccine and oncolytic cancer therapy. Moreover, these findings can provide knowledge for understanding fundamental mechanisms of eukaryotic translation.